Fig 2: There is a single species of caveolar coat complex.(A) Cross-linked and detergent solubilised (1% Triton X-100/1% octyl glucoside) HeLa cell extracts were fractionated by velocity centrifugation (10–40% sucrose), followed by Western blotting of fractions 1–12. Gradients were prepared from cells expressing either GFP (the control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, cavin 3, or GFP. The high molecular weight (HMW) peak of caveolin 1 and cavins is boxed. The strong band in the cavin 3 blot (marked with an asterisk (*); fractions 1–4) are nonspecific: they are also observed in cavin 3 knockout cells and in HeLa cells depleted of cavin 3 by siRNA (see Figure 5A). (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin 1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. Control was GFP-expressing cells. (C) Cavin 1, 2, and-3-GFP were immuno-isolated from pooled gradient HMW fractions 8–10. Control immuno-isolations were performed from pooled fractions 8–10 of cells expressing GFP or flotillin-2-GFP. Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, caveolin 1, EHD2, and pacsin 2. Note the absence of EHD2 and pacsin 2 from the caveolar coat complex. (D) HMW complexes immuno-isolated from cross-linked HeLa cells expressing caveolin-1-GFP, or cavin 1, 2, or-3-GFP were analyzed by LC-MS/MS. The graph is showing the number of peptides identified from caveolin 1 and cavin proteins in each complex. Other proteins identified by mass spectrometry are not shown.

Fig 3: Caveolar proteins are normally not expressed in proximal tubules, but are induced in atrophic tubular segments. Immunohistochemical stainings of human renal tissue showing the distribution of caveolar proteins. Expression of caveolin-1/CAV1 and caveolin-2/CAV2 (a through d) was observed in the PECs, endothelial cells and smooth muscle cells (SMC). No caveolin-3/CAV3 staining could be detected (e and f). PTRF/CAVIN1 staining was observed in PECs and SMCs of large arteries (g and h). PECs and endothelial cells stained positive for SDPR/CAVIN2 (i and j). PRKCDBP/CAVIN3 staining was observed in PECs, and SMCs of arteries, (k and l) whereas MURC/CAVIN4 staining could not be detected (data not shown). In proximal tubules undergoing atrophy due to normal kidney ageing, tubular cells were positive for caveolin-1/CAV1, caveolin-2/CAV2, SDPR/CAVIN2 and PRKCDBP/CAVIN3 (m-p). G - glomerulus, PT - proximal tubules, IC - inflammatory cells, PEC - parietal epithelial cells. Inserted squares mark areas magnified in the following image

Fig 4: Long-term CIH exposure specifically impacts cavin expression. (A–C) mRNA expression of caveolar proteins was determined by quantitative RT-PCR (A) and caveolar protein expression by western-blot (B,C) in epididymal WAT of mice exposed to Normoxia (N) or CIH for 6 weeks. Quantification of relative mRNA expression of Caveolin-1 (Cav-1), Caveolin-2 (Cav-2), Cavin-1, Cavin-2, and Cavin-3 (normalized to GAPDH mRNA expression) is presented in panel (A) (n = 4 mice per condition). Immunoblots of caveolar protein expression are presented in panel (B). The dash line separates normoxic (N) and 6 weeks-CIH samples, run on the same blot, but not loaded adjacently. Quantification of caveolin-1/Cav-1, cavin and EHD2 relative protein expressions (normalized to aP2 protein) are presented in panel (C), as mean ± sem with dot plots representing relative protein signal for each mouse explored. Significant differences between conditions was evaluated by Student t-test, *p < 0.05 vs. normoxic mice.

Fig 5: Cavin 2 and cavin 3 form distinct subcomplexes with cavin 1 and caveolin 1.(A) Gradient fractions (10–40% sucrose) of non-cross-linked and detergent solubilised (1% Triton X-100) HeLa cell extracts were analysed by Western blotting. Gradients were prepared from cells expressing either GFP (as control cells), cavin 1-GFP, cavin 2-GFP, or cavin 3-GFP. Membranes were probed with antibodies against caveolin 1, cavin 1, or GFP. Pooled fractions 3–5 and 6–8 used for immuno-isolation shown in (C) are boxed. (B) Quantification of the distribution of cavin 1, 2, and-3-GFP, cavin 1, and caveolin-1 in velocity gradients as shown in (A). Relative protein amounts were determined by densitometry of Western blots. The data are expressed as an average percentage of protein in each fraction calculated from four independent experiments. (C) Cavin 1, 2, and -3-GFP, flotillin-2-GFP, caveolin-1-GFP, or GFP were immuno-isolated from gradient fractions 3–5 or 6–8 as shown in (A). Eluted proteins were analysed by Western blotting using antibodies against GFP, cavin 1, or caveolin 1. (D) Caveolar coat complexes immuno-isolated from cross-linked HeLa cells stably expressing cavin-2-GFP were incubated with increasing concentrations of DTT to partially reduce crosslinks and analyzed by Western blotting using anti-caveolin 1 or anti-cavin 1 antibodies. Monomeric and oligomeric species of caveolin 1 and cavin 1 are indicated. Note the 180 kDa cavin 1 trimer.